Journal
NATURE BIOTECHNOLOGY
Volume 41, Issue 5, Pages 673-+Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41587-022-01533-6
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Improved cytosine base editors are generated by phage-assisted evolution of a deoxyadenosine deaminase, which exhibit small size, low off-target activity, and high on-target activity. These modified base editors have significant application potential in cell and gene editing.
Cytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination. Compared to commonly used CBEs, TadA-derived cytosine base editors (TadCBEs) offer similar or higher on-target activity, smaller size and substantially lower Cas-independent DNA and RNA off-target editing activity. We also identified a TadA dual base editor (TadDE) that performs equally efficient cytosine and adenine base editing. TadCBEs support single or multiplexed base editing at therapeutically relevant genomic loci in primary human T cells and primary human hematopoietic stem and progenitor cells. TadCBEs expand the utility of CBEs for precision gene editing. Improved cytosine base editors are generated by phage-assisted evolution of a deoxyadenosine deaminase.
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